Fluorescent detection method and reagent

ABSTRACT

Disclosed is a method for increasing the fluorescence of a Cyanine dye molecule comprising at least one NO 2  group characterised by the reduction of the at least one NO 2  group to NHOH or NH 2  by the action of a nitroreductase. The cyanine dye molecule comprising at least one NO 2  group can be used as a substrate for detecting nitroreductase enzyme activity in a composition and allows for the use of a nitroreductase enzyme in an enzyme-reporter system for the detection of analytes, binding reaction or gene expression.

[0001] The present invention relates to methods for generating moleculeswith a high fluorescence output from molecules with a lower ornegligible fluorescence and which may be catalysed by enzymatic action.In particular, the invention relates to methods for achievingfluorescence detection of analytes by using enzymatic means to generatefluorescent reporter molecules.

[0002] The use of fluorescence as a detection modality in biologicalassays is widespread, and a diverse variety of procedures are availableto generate fluorescence in assay conditions for detection by a widerange of techniques. These techniques include fluorescence microscopy,fluorescence immunoassay and flow cytometry.

[0003] Among the methods used to generate a fluorescent signal are thosewhich use an enzyme to convert a non-fluorescent substrate to afluorescent product.

[0004] The enzyme may be coupled to an assay component, for example toan antibody in an immunoassay or to a nucleic acid molecule in a nucleicacid hybridisation assay, and is typically used to generate afluorescence signal from a non-fluorescent substrate. In such methods,the fluorescence intensity of the product provides the assay signal, andcorrelates with the amount of analyte in the assay (e.g. an antigen inan immunoassay or a complementary nucleic acid sequence in a nucleicacid hybridisation assay). Examples of such enzyme based fluorescencemethod assays are described in “Applications of Fluorescence inImmunoassays”, Chapter 9, pages 223-232, I. A. Hemmila, John Wiley &Sons, New York, 1991 (immunoassays) and “Nonisotopic DNA ProbeTechniques”, Chapter 1, pages 3-23, L. J. Kricka, Academic Press Inc.,New York, 1992 (nucleic acid hybridisation assays) and enzymes used insuch assays include Horse Radish Peroxidase (HRP) and alkalinephosphatase.

[0005] Alternatively, the enzyme may be generated by protein synthesisin the course of the assay. For example, in an in vivo gene expressionassay the enzyme is synthesised from a gene, usually termed a reportergene, which is inserted into a cell or organism in which it does notoccur naturally or is only found at low levels, in such a way thatexpression of the reporter gene is linked to the expression of acellular gene of interest. Consequent processing of the enzyme'snon-fluorescent substrate to a fluorescent product correlates with theexpression of the reporter gene, and hence provides an indirect measureof the expression of the cellular gene of interest. Enzymes that aresynthesised from reporter genes and currently used in fluorescent assaysfor the measurement of in vivo gene expression include β-galactosidase,alkaline phosphatase and β-lactamase.

[0006] β-galactosidase is a bacterial enzyme and has been wellcharacterised for use in such enzyme-reporter assays in combination withsubstrates which can be rendered fluorescent by enzyme activity. Thedetection of β-galactosidase expression is described in “FluorescenceMicroscopy and Fluorescent Probes”, pages 211-215, J. Slavik, PlenumPress, London, 1996. Here, a non-fluorescent substrate, CMFDG, ismicroinjected into cells expressing the enzyme where it is hydrolysedinto a fluorescent form giving a measure of enzyme activity. However, asmammalian cells display some endogenous β-galactosidase activity, somebackground fluorescence of the converted substrate is observed in invivo assays for gene expression even in the absence of signals leadingto the expression of β-galactosidase from the reporter gene.

[0007] Mammalian cells can also have some endogenous alkalinephosphatase enzyme activity again leading to a background of activationof a non-fluorescent substrate into its fluorescent form in in vivoassays based on alkaline phosphatase expression. In this case, thefluorescent substrate is, typically, fluorescein diphosphate. Inaddition, optimal alkaline phosphatase activity requires pH 9.8 whichlimits the use of this enzyme in in situ assays.

[0008] More recently, a fluorescent substrate for β-lactamase has beendocumented (see U.S. Pat. No. 5,955,604, Tsien et al.). The substratedescribed therein, CCF2, is suitable for use in a FRET based assay (asdescribed below) in which the donor and acceptor molecules in thefluorescent substrate are separated by the enzyme activity ofβ-lactamase to generate a fluorescent signal from the donor molecule.

[0009] However, the fluorochromes used in these techniques are,typically, fluorescein and its derivatives (Handbook of FluorescentProbes and Research Chemicals, Molecular Probes, 6^(th) Edition, 1996 orsee www.probes.com). Fluorescein-based fluorochromes typically haveexcitation wavelengths in the WV to blue region of the spectrum andemission wavelengths in the blue to green region of the spectrum (i.e.excitation at approx. 488 nm and emission at approx. 510 nm). Thesecharacteristics confer certain restrictions on the use of thesefluorochromes with biological materials as these wavelengths coincidewith the excitation and emission ranges of a number of biologicalmolecules. This can give high background fluorescence in biologicalassays with a resultant reduced sensitivity of detection.

[0010] Moreover, as all these known techniques yield signals within thesame region of the spectrum, there is limited scope for multiplesignalling systems being employed and read simultaneously.

[0011] Dyes based on fluorescein have a number of other disadvantages,including their tendency to photobleach when illuminated by strongexcitation sources. Furthermore, some products from enzyme substratesbased on these fluorochromes are pH sensitive, which can lead tovariation in fluorescence in different environments. Problems may ariseusing these fluorochromes in intracellular assays as excitation in theUV region, requiring high energy excitation at a short wavelength, maygive rise to cell damage which can, in turn, lead to misleading results.

[0012] To overcome these shortcomings of existing enzyme fluorescencesubstrates, there is a requirement for a molecule (or molecules) whichis the substrate for a non-ubiquitous enzyme and which has zero or lowfluorescence in a first form and which, on reaction with the enzyme,yields an environmentally stable fluorescent product which can emitlight over a broad range of the spectrum. The range of emission couldbe, for example in the range of 500-900 nm region of the spectrum.

[0013] The Cyanine dyes (sometimes referred to as “Cy dye™”), described,for example, in U.S. Pat. No. 5,268,486, are a series of biologicallycompatible fluorophores which are characterised by high fluorescenceemission, environmental stability and a range of emission wavelengthsextending into the near infra-red which can be selected by varying theinternal molecular skeleton of the fluorophore.

[0014] Recently, cyanine dyes have been developed for use inFluorescence Resonance Energy Transfer (FRET) assays. The principal ofFRET was described in U.S. Pat. No. 4,996,143 and, more recently, inPCT/GB99/01746 (publication number WO99/64519). Briefly, FRET assaysdepend on an interaction between two fluorophores, a donor fluorophoreand an acceptor fluorophore. When the donor and acceptor molecules arein close enough proximity, the fluorescence of the donor molecule istransferred to the acceptor molecule with a resultant decrease in thelifetime and a quenching of fluorescence of the donor species and aconcomitant increase in the fluorescence intensity of the acceptorspecies. The use of FRET labels in biological systems is well known. Theprinciple has been used in the detection of binding events or cleavagereactions in assays which employ FRET. In the case of peptide cleavagereactions, a fluorescent donor molecule and a fluorescent acceptormolecule are attached to a peptide substrate on either side of thepeptide bond to be cleaved and at such a distance that energy transfertakes place. A peptide cleavage reaction will separate the donor andacceptor molecules and thus the fluorescence of the donor molecule willbe restored.

[0015] In one format of this principle, a fluorescent moiety is causedto be in close proximity with a “quencher” molecule such that the energyfrom the excited donor fluorophore is transferred to the quencher anddissipated as heat rather than fluorescence energy. In this case,residual fluorescence is minimised when the two components of thedonor-quencher pair are in close proximity and a large change in signalcan be obtained when they are separated.

[0016] Cyanine dyes suitable for use as acceptor or “quencher” moleculesin a FRET assay have been developed (see PCT/GB99/01746) by makingcertain modifications to cyanine dyes through introduction of chemicalgroups which have the effect of diminishing or abolishing thefluorescence of the molecule. One example of such a chemicalmodification is the introduction of-NO₂ groups. Such quenched Cy dyesare referred to as Cy-Q dyes or “dark dyes”.

[0017] The bacterial enzymes termed nitroreductases have been shown tocatalyse the general reaction set out below in reaction scheme 1:

[0018] where, in the presence of NADH or NADPH, one or more —NO₂ groupson an organic molecule are reduced to a hydroxylamine group which maysubsequently be converted to an amine group.

[0019] Bacterial nitroreductases have been used, in anti-tumour therapy,to convert prodrug molecules into their corresponding cytotoxic forms(Anlezark et al 1995, Biochem-Pharmacol 50(5), 609-18) by removing orreducing one or more —NO₂ groups on the prodrug substrate. Suchsubstrates include p-nitrobenzyloxycarbonyl derivatives of cytotoxiccompounds. Selective killing of tumour cells can be achieved bytargeting expression of the nitroreductase gene to tumour cells andadministering the prodrug to the affected tissue (as described, forexample, in U.S. Pat. No. 5,780,585). The prodrug substrate is convertedto its cytotoxic form in the tumour cells expressing nitroreductasewhile the surrounding cells (which do not contain the nitroreductase)remain unaffected.

[0020] Nitroreductases have also been used in bio-remediation processesfor clearing nitroaromatic compounds which may create environmental orhealth hazards. U.S. Pat. No. 5,777,190 describes a catalytic methodinvolving oxygen-sensitive nitroreductases for reducing nitroaromaticcompounds which may be controlled, to prevent the reaction fromprogressing to completion, by the addition of oxygen. Suggestedsubstrates include nitrobenzene, trinitrotoluene andorthochloronitrobenzene with preferred oxygen sensitive nitroreductaseenzymes including ferredoxin NADP, xanthine oxidase and glutathionereductase.

[0021] To date, there appear to be no reports that an NO₂-containingmodified cyanine dye molecule could act as a substrate for anitroreductase to generate a fluorescent molecule.

[0022] The present invention provides a method for increasingfluorescence of a modified cyanine dye comprising at least one NO₂(nitro) group, for example, a Cy-Q dye. This can be achieved byenzymatic conversion of an NO₂ group in such a Cy-Q dye to a NHOH or NH₂by the action of a nitroreductase (NTR). Depending on the structure ofthe Cy-Q dye, the fluorescence emission from the product of the Cy-Q/NTRreaction may occur across a wide range of wavelengths, typically 500-900nm, in contrast to existing reporters which emit only in the blue-greenregion of the spectrum. This emission at longer wavelengths isadvantageous in avoiding background fluorescence and increasingsensitivity in biological systems.

[0023] Moreover, the fluorescence emission characteristics of theCy-Q/NTR reaction product can be altered to suit the application bymaking changes to the internal structure of the Cy-Q molecule, withoutchanging the extremities of the molecule, e.g. the NO₂ groups, that areinvolved in reaction with nitroreductase. Thus, fluorescent reporterscompatible for use with other fluors in multiplex systems can beprovided.

[0024] In addition, the structure-defined emission characteristics ofthe Cy-Q make it suitable for inclusion in a paired fluorophoreratiometric reporter molecule where one member of the pair is afluorescent dye and the second member is a Cy-Q dye. Nitroreductaseaction on the Cy-Q leads to a change in the ratio of fluorescenceemission from the two fluors when excited and monitored at two differentwavelengths. Such a ratiometric reporter molecule allows measurement ofenzyme activity to be made independent of the concentration of thereporter molecule.

[0025] The invention accordingly provides in a first aspect, a methodfor increasing the fluorescence of a dye molecule comprising at leastone NO₂ group characterised by the reduction of said at least one NO₂group to NHOH or NH₂.

[0026] Suitable NO₂-containing dyes include fluorescent dyes such ascyanine derivates, Cy-Q (described in PCT/GB99/017460), NO₂-lanthanidechelates (described, for example, in Latra, M. J., Lumin. 1997,75,149-169 and Blasse, G., J. Phys. Chem. 1998; 92; 2419-2422) andNO₂-containing fluoresceins, pyrenes, rhodamines, coumarins or BODIPY™dyes.

[0027] In one embodiment of the first aspect, the dye moleculecomprising at least one NO₂ group for use in the present invention is amodified cyanine dye compound having the Formula I:

[0028] wherein groups R³, R⁴, R⁵ and R⁶ are attached to the ringscontaining X and Y or, optionally, are attached to atoms of the Z¹ andZ² ring structures and n is an integer from 1-3;

[0029] Z¹ and Z² each represent a bond or the atoms necessary tocomplete one or two fused aromatic rings each ring having five or sixatoms, selected from carbon atoms and, optionally, no more than twooxygen, nitrogen and sulphur atoms;

[0030] X and Y are the same or different and are selected from bis-C₁-C₄alkyl- and C₄-C₅ spiro alkyl-substituted carbon, oxygen, sulphur,selenium, —CH═CH— and N—W wherein N is nitrogen and W is selected fromhydrogen, a group —(CH₂)_(n)R⁸ where m is an integer from 1 to 26 and R⁸is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano,aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate,phosphonate, polyethylene glycol, substituted amino, quaternaryammonium, nitro, primary amide, substituted amide, and groups reactivewith amino, hydroxyl, carbonyl, carboxyl, phosphoryl, and sulphydrylgroups;

[0031] groups R³, R⁴, R⁵, R⁶ and R⁷ are independently selected from thegroup consisting of hydrogen, substituted or unsubstituted C₁-C₄ alkyl,OR⁹, COOR⁹, nitro, amino, acylamino, quaternary ammonium, phosphate,sulphonate and sulphate, where R⁹ is substituted or unsubstituted andselected from H, C₁-C₄ alkyl, amino, aldehyde, acetal, ketal, halo,cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate,substituted amino, quaternary ammonium, nitro, primary amide,substituted amide, and groups reactive with amino, hydroxyl, carbonyl,carboxyl, phosphoryl, and sulphydryl groups.

[0032] groups R¹ and R² are selected from C₁-C₁₀ alkyl which may beunsubstituted or substituted;

[0033] characterised in that at least one of the groups R¹, R², R³, R⁴,R⁵, R⁶ and R⁷ comprises at least one nitro group which reduces thefluorescence emission of said dye such that it is essentiallynon-fluorescent.

[0034] Suitably, the at least one nitro group comprised in the dyes ofFormula I may be attached directly to the rings containing X and Y. Inthe alternative, a mono- or di-nitro-substituted benzyl group may beattached to the rings containing X and Y, which optionally may befurther substituted with one or more nitro groups attached directly tothe aromatic rings.

[0035] In one embodiment, R¹ and R² may be selected from C₁-C₁₀ alkylwhich may be substituted with groups including NH₂, OH, COOH, SO₃H,PO₄H, SH, polyethylene glycol and phenyl. Where phenyl is substituted,it may optionally be substituted by up to two substituents selected fromcarboxyl, sulphonate and nitro groups.

[0036] Examples of the non-fluorescent cyanine dye molecules suitablefor use in the present invention have either Formula II and Formula III:

[0037] Especially preferred are NO₂-containing “dark dye” forms of anyof the following Cy dyes. The excitation (Abs) and emission (Em)characteristics of the unmodified dye molecules are shown: DyeFluorescence Colour Abs (nm) Em (nm) Cy2 Green 489 506 Cy3 Orange 550570 Cy3.5 Scarlet 581 596 Cy5 Far red 649 670 Cy5.5 Near-IR 675 694 Cy7Near-IR 743 767

[0038] In another embodiment of the first aspect, the reduction of a NO₂group is catalysed by an enzyme, which can be a nitroreductase and,preferably, a bacterial nitroreductase.

[0039] Importantly, this means that cyanine-based dyes can be used insuch an enzyme-substrate reaction at the same time as any of theconventional enzyme-substrate reactions which give a fluorescencereadout in the blue/green region of the spectrum. Measurements can bemade simultaneously using two different wavelengths; for example,fluorescein-based molecules could be detected at Abs 488/Em 510 whereasreduced cyanine-based molecules, such as those based on Cy5, could bedetected at Abs 649/Em 670. This would allow multiplexing i.e. measuringa number of different in vitro or in vivo effects simultaneously.

[0040] NO₂-containing cyanine molecules can be described as“non-fluorescent dyes” i.e. those dyes which have an intrinsic lowefficiency for converting absorbed incident light into fluorescence. Theeffectiveness of a non-fluorescent dye as a nitroreductase substrate isthe extent to which it can convert incident light to fluorescence aftera reduction reaction.

[0041] An increase in fluorescence can be measured relative to a controlsample comprising an NO₂-containing cyanine dye molecule substrate inthe absence of a nitroreductase enzyme.

[0042] In a second aspect of the invention there is provided a methodfor detecting nitroreductase enzyme activity in a compositioncomprising:

[0043] a) mixing said composition with a dye compound comprising atleast one NO₂ group under conditions to promote nitroreductase activity;and

[0044] b) measuring an increase in fluorescence wherein said increase isa measure of the amount of nitroreductase activity.

[0045] In a particularly preferred embodiment, the dye compound is acyanine dye compound comprising at least one NO₂ group.

[0046] Suitably the cyanine dye compound is a compound of Formula I ashereinbefore described.

[0047] In one embodiment of the second aspect, the composition comprisesa cell or cell extract. In principle, any type of cell can be used i.e.prokaryotic or eukaryotic (including bacterial, mammalian and plantcells). Where appropriate, a cell extract can be prepared from a cell,using standard methods known to those skilled in the art (MolecularCloning, A Laboratory Manual 2^(nd) Edition, Cold Spring HarbourLaboratory Press 1989), prior to measuring fluorescence.

[0048] Typical conditions for nitroreductase activity compriseincubation of the composition and a cyanine dye molecule comprising atleast one NO₂ group at approximately 37° C. in the presence of NADH andFMN.

[0049] In a third aspect of the invention there is provided a method ofdetecting analytes comprising;

[0050] a) providing a nitroreductase enzyme coupled to an assay reagentunder conditions where the amount of activity of the enzyme isproportional to the amount of analyte in the assay;

[0051] b) providing a dye compound comprising at least one NO₂ group;and

[0052] c) measuring an increase in fluorescence as a measure of theamount of nitroreductase activity.

[0053] In a fourth aspect of the invention there is provided an assaymethod which comprises:

[0054] a) binding a first component of a specific binding pair to asurface;

[0055] b) adding a second component of the specific binding pair underconditions to promote binding between the components, said secondcomponent being labelled with a nitroreductase enzyme;

[0056] c) adding a dye compound comprising at least one NO₂ group underconditions suitable for nitroreductase activity; and

[0057] d) detecting binding of the second component to the firstcomponent by measuring an increase in fluorescence as a measure of boundnitroreductase activity.

[0058] In a particularly preferred embodiment of the third or fourthaspects of the invention, the dye compound is a cyanine dye compoundcomprising at least one NO₂ group.

[0059] Suitably, the dye compound employed in the third or fourthaspects is a cyanine dye compound of Formula I as hereinbeforedescribed.

[0060] In one embodiment of the fourth aspect, said specific bindingpair is selected from the group consisting of antibodies/antigens,lectins/glycoproteins, biotin/streptavidin, hormone/receptor,enzyme/substrate, DNA/DNA, DNA/RNA, DNA/binding protein or engineeredbinding partner.

[0061] Briefly, an in vitro assay method for the detection of antibodybinding may be configured as follows. An antibody specific for anantigen of interest may be labelled by covalently linking it to anenzymatically active nitroreductase. Said labelled antibody can then beintroduced into the test sample containing the antigen under bindingconditions. After washing to remove any unbound antibody, the amount ofantibody bound is detected by incubating the sample with thenon-fluorescent cyanine dye substrate under conditions fornitroreductase activity and measuring an increase in fluorescence. Theamount of fluorescence detected will be proportional to the amount ofnitroreductase-labelled antibody that has bound to the analyte.

[0062] In an in vitro assay for detecting the binding of nucleic acidsby hybridisation, either of the pair of target and probe nucleic acid isbound to a membrane or surface. The unbound partner is labelled withnitroreductase and incubated under hybridising conditions with the boundnucleic acid. Unbound, labelled nucleic acid is washed off and theamount of bound, labelled nucleic acid is measured by incubating themembrane or surface with a non-fluorescent cyanine dye under conditionssuitable for nitroreductase activity. The amount of increase influorescence gives a measure of the amount of bound labelled DNA.

[0063] Methods for coupling enzymes to other biomolecules, e.g. proteinsand nucleic acids, are well known. (Bioconjugate Techniques, AcademicPress 1996). Coupling may be achieved by direct means, for example byuse of a suitable bifunctional crosslinking agent (e.g.N-[β-Maleimidopropionic acid]hydrazine, Pierce) to covalently link theenzyme and binding partner. Alternatively, coupling may be achieved byindirect means, for example by separately biotinylating the enzyme andthe binding partner using a chemically reactive biotin derivative, (e.g.N-hydroxysuccinimido-biotin, Pierce) and subsequently coupling themolecules through a streptavidin bridging molecule.

[0064] Cell based assays are increasingly attractive over in vitrobiochemical assays for use in high throughput screening (HTS). This isbecause cell based assays require minimal manipulation and the readoutscan be examined in a biological context that more faithfully mimics thenormal physiological situation. Such in vivo assays require an abilityto measure a cellular process and a means to measure its output. Forexample, a change in the pattern of transcription of a number of genescan be induced by cellular signals triggered, for example, by theinteraction of an agonist with its cell surface receptor or by internalcellular events such as DNA damage. The induced changes in transcriptioncan be identified by fusing a reporter gene to a promoter region whichis known to be responsive to the specific activation signal.

[0065] In fluorescence-based enzyme-substrate systems, an increase influorescence gives a measure of the activation of the expression of thereporter gene.

[0066] Accordingly, in a fifth aspect of the invention, there isprovided an assay method which comprises:

[0067] a) contacting a host cell with a dye compound comprising at leastone NO₂ group, wherein said host cell has been transfected with anucleic acid molecule comprising expression control sequences operablylinked to a sequence encoding a nitroreductase; and

[0068] b) measuring an increase in fluorescence as a measure ofnitroreductase gene expression.

[0069] In a particularly preferred embodiment, the dye compound is acyanine dye compound comprising at least one NO₂ group. Suitably, thecyanine dye is a compound of Formula I as hereinbefore described.

[0070] Methods for using a variety of enzyme genes as reporter gene inmammalian cells are well known (for review see Naylor L. H. (1999)Biochemical Pharmacology 58, 749-757). The reporter gene is chosen toallow the product of the gene to be measurable in the presence of othercellular proteins and is introduced into the cell under the control of achosen regulatory sequence which is responsive to changes in geneexpression in the host cell. Typical regulatory sequences include thoseresponsive to hormones, second messengers and other cellular control andsignalling factors. For example, agonist binding to seven transmembranereceptors is known to modulate promoter elements including the cAMPresponsive element, NFAT, SRE and AP1; MAP kinase activation leads tomodulation of SRE leading to Fos and Jun transcription; DNA damage leadsto activation of transcription of DNA repair enzymes and the tumoursuppressor gene p53. By selection of an appropriate regulatory sequencethe reporter gene can be used to assay the effect of added agents orcellular processes involving the chosen regulatory sequence under study.

[0071] For use as a reporter gene, the nitroreductase gene may beisolated by common methods, for example by amplification from a cDNAlibrary by use of the polymerase chain reaction (PCR) nd (MolecularCloning, A Laboratory Manual 2^(nd) Edition, Cold Spring HarbourLaboratory Press 1989 pp 14.5-14.20). Once isolated, the nitroreductasegene may be inserted into a vector suitable for use with mammalianpromoters (Molecular Cloning, A Laboratory Manual 2^(nd) Edition, ColdSpring Harbour Laboratory Press 1989 pp 16.56-16.57) in conjunction withand under the control of the gene regulatory sequence under study. Thevector containing the nitroreductase reporter and associated regulatorysequences may then be introduced into the host cell by transfectionusing well known techniques, for example by use of DEAE-Dextran orCalcium Phosphate (Molecular Cloning, A Laboratory Manual 2^(nd)Edition, Cold Spring Harbour Laboratory Press 1989 pp 16.30-16.46).Other suitable techniques will be well known to those skilled in theart.

[0072] Nitroreductase has been shown to be retained in cells whenexpressed in this manner (see Bridgewater et al. Eur. J. Cancer 31 a,2362-70).

[0073] In a preferred embodiment of the fifth aspect of the invention,the cyanine dye molecule comprising at least one NO₂ group is permeableto cells. Preferably, at least one of groups R¹, R², R³, R⁴, R⁵, R⁶ andR⁷ of the cyanine dye molecule of Formula I comprises a cell membranepermeabilising group. Membrane permeant compounds can be generated bymasking hydrophilic groups to provide more hydrophobic compounds. Themasking groups can be designed to be cleaved from the fluorogenicsubstrate within the cell to generate the derived substrateintracellularly. Because the substrate is more hydrophilic than themembrane permeant derivative it is then trapped in the cell. Suitablecell membrane permeabilising groups may be selected from acetoxymethylester which is readily cleaved by endogenous mammalian intracellularesterases (Jansen, A. B. A. and Russell, T. J., J. Chem Soc. 2127-2132(1965) and Daehne W. et al. J. Med-. Chem. 13, 697-612 (1970)) andpivaloyl ester (Madhu et al., J. Ocul. Pharmacol. Ther. 1998, 14, 5, pp389-399) although other suitable groups including delivery molecules(such as delivery peptides) will be recognised by those skilled in theart.

[0074] In a sixth aspect of the invention there is provided anNO₂-containing compound in accordance with Formula I wherein saidcompound has been modified so as to be capable of entering a cell.Accordingly, in a particularly preferred embodiment, there is providedan NO₂-containing compound selected from compounds of Formula IIIa orFormula IIIb.

[0075] Typically, to assay the activity of an agent to activate cellularresponses via the regulatory sequence under study, cells transfectedwith the nitroreductase reporter are incubated with the test agent,followed by addition of a cell-permeant cyanine dye substrate, such as acyanine dye molecule comprising at least one NO₂ group. After anappropriate period required for conversion of the cyanine dye substrateto a form showing higher fluorescence, the fluorescence emission fromthe cells is measured at a wavelength appropriate for the chosen cyaninedye. For example, for the compound Cy-Q F (shown in FIG. 1a),fluorescence emission would be monitored at 690 nm with excitation at650 nm. Measurement of fluorescence maybe readily achieved by use of arange of detection instruments including fluorescence microscopes (e.g.LSM 410, Zeiss), microplate readers (e.g. CytoFluor 4000, Perkin Elmer),CCD imaging systems (e.g. LEADseeker™, Amersham Pharmacia Biotech) andFlow Cytometers (e.g. FACScalibur, Becton Dickinson).

[0076] The measured fluorescence is compared with fluorescence fromcontrol cells not exposed to the test agent and the effects, if any, ofthe test agent on gene expression modulated through the regulatorysequence is determined from the ratio of fluorescence in the test cellsto the fluorescence in the control cells.

[0077] Where appropriate, a cell extract can be prepared usingconventional methods.

[0078] Accordingly, in a seventh aspect of the invention, there isprovided an assay method which comprises:

[0079] a) contacting a host cell extract with a dye compound comprisingat least one NO₂ group wherein said host cell has been transfected witha nucleic acid molecule comprising expression control sequences operablylinked to a sequence encoding a nitroreductase; and

[0080] b) measuring an increase in fluorescence as a measure ofnitroreductase gene expression.

[0081] In a particularly preferred embodiment, the dye compound is acyanine dye compound comprising at least one NO₂ group. Suitably, thecyanine dye is a compound of Formula I as hereinbefore described.

[0082] In one embodiment of any of the previous aspects of theinvention, increased fluorescence of the cyanine dye molecule isidentified by analysis of fluorescence emission in the range 500 to 900nm, preferably 550-780 nm, and, most preferably 665-725 nm.

[0083] In an eighth aspect of the invention there is provided a kit fora reporter system comprising a means for expressing a nitroreductaseenzyme and a dye molecule comprising at least one NO₂ group.

[0084] Suitable means for expressing a nitroreductase enzyme include anexpression plasmid or other expression construct. Methods for preparingsuch expression constructs are well known to those skilled in the art.

[0085] In a ninth aspect of the invention there is provided a kit fordetecting the presence of one component of a specific binding paircomprising a nitroreductase enzyme coupled to the other component ofsaid specific binding pair and a dye molecule comprising at least oneNO₂ group.

[0086] In a prefered embodiment of the eighth or ninth aspect, the dyemolecule is a cyanine dye molecule comprising at least one NO₂.Suitably, the cyanine dye is a compound of Formula I as hereinbeforedescribed.

[0087] The change in fluorescence which arises from nitroreductaseaction on dye molecules comprising at least one NO₂, and particularlyCy-Q dyes, can be exploited in the construction of ratiometricfluorescence reporters or “cassettes” based on linked fluorophores oneof which is a molecule such as a Cy-Q dye.

[0088] Accordingly, in a tenth aspect of the invention there is provideda paired fluorophore ratiometric reporter of Formula IV:

[0089] wherein:

[0090] D₁ is a detectable fluorophore;

[0091] D₂ is a cyanine dye molecule having Formula (I); and

[0092] L is a linker group.

[0093] Suitably, D₁ and D₂ are selected such that excitation of thecassette is at two different wavelengths, λ1 and λ2, where thewavelengths are chosen to be suitable to elicit fluorescence emissionfrom the fluorophore D₁ (at wavelength λ3) and the fluorophorecorresponding to D₂ (at wavelength λ4), and from which D₂ is derived,yet lacking the at least one NO₂ group.

[0094] In a particularly preferred embodiment, D₂ is selected from Cy3Qand Cy5Q.

[0095] In a preferred embodiment, L is a cleavable linker, for example,chemically cleavable, photocleavable (e.g. nitrobenzylalcohol) orenzymatically cleavable (e.g. ester, amide, phosphodiester, azo) byenzymes such as proteases. Suitable methods for cleaving such a linkerare well known and described, for example, in Gerard Marriott et al.,Preparation and photoactivation of caged fluorophores and caged proteinsusing a new cross-linking reagent, Bioconjugate Chemistry; (1998); 9(2);143-151 and WO 00/75358.

[0096] In one embodiment, D₁ and D₂ may be in a FRET arrangement.

[0097] Preferably, the compound of Formula IV is rendered cell ormembrane permeable.

[0098] In a particularly preferred embodiment of the tenth aspect, thereis provided a compound of Formula V.

[0099] In this embodiment, D₁ is 8-hydroxy-pyrene-1,3,6-trisulfonic acid(Cascade Blue™), D₂ is Cy5Q and L is an ester linkage.

[0100] In this embodiment, D₁ and D₂ are arranged in a FRET arrangementsuch that the fluorescence of D₁ is quenched by D₂. When the cassette ofFormula V is incubated in the presence of intracellular enzymes (e.g.when the cassette is successfully transported into a cell) the linker,L, is cleaved thus releasing D₁ from the energy transfer arrangementand, when excited at wavelength XI, fluorescence emission at wavelengthλ3 can be detected. Under these conditions determination of thefluorescence emission at λ3 and comparison with the unreacted cassettewill give a measure of the cleavage of the linker moiety, L (and, thus,a measure of uptake of the cassette into cells, for example). In theabsence of nitroreductase, only low or zero emission from D₂ atwavelength λ4 will be detected. In the presence of nitroreductase theCyQ moiety is reduced to form a fluorescent form of Cy such thatexcitation at wavelength λ2 will give emission at wavelength λ4. Underthese conditions, determination of the fluorescence emission at λ4 andcomparison with the fluorescence emission at λ4 of the unreactedcassette will give a measure of the degree of conversion of D₂ into itsreduced form and hence a measure of nitroreductase activity.

[0101] Suitably, the compound of Formula IV may be used in an assaymethod in accordance with any one of the third, fourth, fifth, sixth oreighth aspects of the invention.

SPECIFIC DESCRIPTION

[0102] For the purposes of clarity, certain embodiments of the presentinvention will now be described by way of example with reference to thefollowing figures:

[0103]FIG. 1a shows the chemical structure of the compound of FormulaII, termed Cy-Q F.

[0104]FIG. 1b shows a fluorescence emission spectrum of Cy-Q F incubatedfor various times in the presence of E. coli B nitroreductase.

[0105]FIG. 2a shows the chemical structure of the compound of FormulaIII, termed Cy-Q G.

[0106]FIG. 2b shows a fluorescence emission spectrum of Cy-Q G incubatedfor various times in the presence of E. coli B nitroreductase.

[0107]FIG. 3a shows HPLC analysis of Cy-Q G.

[0108]FIG. 3b shows HPLC analysis of Cy-Q G treated with nitroreductase.

[0109]FIG. 4a shows MALDI-TOF Mass Spectrometry analysis of Cy-Q G.

[0110]FIG. 4b shows MALDI-TOF Mass Spectrometry analysis of Cy-Q Gtreated with nitroreductase.

[0111]FIG. 5 shows infra-red absorbtion spectroscopy of Cy-Q G and Cy-QG treated with nitroreductase.

[0112]FIG. 6 shows the chemical structure of Cy5Q.

[0113]FIG. 7 shows fluorescence emission different concentration of Cy5Qin the presence nitroreductase.

[0114]FIG. 8 shows the results of an in vitro binding assay withnitroreductase.

[0115]FIG. 9 shows detection of nitroreductase activity in cell lysatesfrom transfected and control cells by detecting Cy5 fluorescence.

[0116]FIG. 10 shows Cy3 fluorescence in cells expressing nitroreductaseand control cells.

[0117]FIG. 11 shows Cy5 fluorescence in cells expressing nitroreductaseand control cells.

[0118]FIG. 12 shows measurement of cellular nitroreductase activity byflow cytometry.

[0119]FIG. 13 shows fluorescence emission spectrum of Cy5Q-cascade bluecassette, a=1 at 649 nm in water, then diluted 100 microlitres+3 mlwater, excitation 450 nm.

[0120]FIG. 14 shows fluorescence emission spectrum of8-hydroxy-pyrene-1,3,6-trisulfonic acid a=0.2 at 455 nm in water, thendiluted 100 microlitres+3 ml water, excitation 450 nm.

[0121]FIG. 15 shows Cy5Q-cascade blue cassette after treatment with NaOHsolution, a=at 650 nm in water, then diluted 100 microlitres+3 ml water,excitation 450 nm.

[0122]FIG. 16 shows relative fluorescence of Cy5Q-cascade blue cassettein the presence of absence of lysate/NTR.

EXAMPLE 1

[0123] A reaction mixture was prepared containing 0.8 mg/ml E. coli Bnitroreductase, 1 mM NADH, 0.4 μm FMN and 0.05 mM Cyanine-Q F (Cy-Q F,FIG. 1a) in phosphate buffered saline on ice. A sample was removedimmediately for analysis by fluorescence spectroscopy, and furthersamples were removed for analysis after incubation of the reactionmixture at 37° C. for 1, 2, and 5 hours.

[0124] Analysis of fluorescence emission in the range 665 nm to 725 nmarising from excitation at 650 nm revealed a marked increase influorescence with time of incubation of Cy-Q F with nitroreductase (FIG.1b), with a maximum emission at 690 nm.

EXAMPLE 2

[0125] A reaction mixture was prepared containing 0.8 mg/ml E. coli Bnitroreductase, 1 mM NADH, 0.4 μm FMN, and 0.05 mM Cyanine-Q F (Cy-Q G,FIG. 2a) in phosphate buffered saline on ice. A sample was removedimmediately for analysis by fluorescence spectroscopy, and furthersamples were removed for analysis after incubation of the reactionmixture at 37° C. for 1, 2, and 5 hours.

[0126] Analysis of fluorescence emission in the range 660 nm to 720 nmarising from excitation at 650 nm revealed a marked increase influorescence with time of incubation of Cy-Q G with nitroreductase (FIG.2b), with a maximum emission at 675 nm.

[0127] Samples of Cy-Q G incubated in the presence and absence ofnitroreductase were analysed by HPLC. The HPLC results (FIGS. 3a and 3b) indicate that nitroreductase treatment of the Cy-Q gives >95%conversion of the Cy-Q G to a product with an HPLC retention time of30.5 minutes, compared to a retention time of 43.2 minutes for thestarting material. The major peaks from the HPLC analysis were subjectedto further analysis by MALDI-TOF mass spectrometry (FIGS. 4a and 4 b).This analysis showed a principal mass of 620.3 for the reaction product,compared to 650.1 for the Cy-Q G starting material, this mass change isconsistent with the conversion of a —NO₂ group to a —NH₂ group, and isconsistent with the proposed enzyme reaction mechanism. This wasconfirmed by further analysis using infra-red absorbtion spectroscopy(FIG. 5) which showed the disappearance of a strong N═O bond absorbtionband on enzyme treatment, consistent with the enzymatic reduction of theCy-Q G —NO₂ group to an amine group.

EXAMPLE 3

[0128] Nitroreductase K_(m) Measurement for Cy5Q

[0129]E. coli B nitroreductase (500 ng) was incubated in the presence ofincreasing concentrations of Cy5Q (FIG. 6) in 200 μl of 10 mM Tris.HClpH 7.5 containing 1 mM NADH and incubated at room temperature for 30minutes.

[0130] Fluorescence from each reaction was measured in a CytoStar(PerSeptive Biosystems) plate reader using 610/20 nm excitation and670/40 nm emission filters. Results were corrected for fluorescence inthe absence of nitroreductase enzyme (FIG. 7) and a Km value of 8.6±0.7μM was calculated using curve fitting software (Prism).

EXAMPLE 4

[0131] In-vitro Binding Assay with Nitroreductase

[0132] Nitroreductase (400 ug, 16.7 nmol) was labelled with 334 nmolSulpho-NHS-biotin (Pierce) in PBS pH 8.0 for 2 hours on ice. Free biotinwas removed by dialysis overnight against PBS pH 7.4 at 4° C.

[0133] Increasing concentrations of biotin (Sigma) from 0-100 nmol/wellwere added to duplicate wells of a streptavidin-coated microtitre plate(Pierce) in 100 ul PBS pH 7.4, followed by 100 ul of PBS containing 0.5ug biotinylated nitroreductase, and the plate incubated for 1 hour atroom temperature.

[0134] Following incubation the plate was washed three times with PBS toremove unbound enzyme and 100 ul of 5 uM Cy5Q in PBS containing 1 mMNADH was added to all wells.

[0135] After incubation for 35 minutes at room temperature to allowreaction between bound nitroreductase and the added Cy5Q the plate wasanalysed on a Cytofluor plate reader (Perseptive) using a 610/20 nmexcitation filter and a 670/40 nm emission filter.

[0136]FIG. 8 shows high fluorescence of Cy5 in the presence of boundnitroreductase. Binding of nitroreductase to the plate was displaced inhigh concentrations of free biotin thus Cy5Q was not reduced and showslow fluorescence.

EXAMPLE 5

[0137] Transfection of Nitroreductase and Measurement of Activity inCell Lysates.

[0138] The E. coli nitroreductase B gene was cloned into the p-Targetmammalian expression vector (Promega) under the control of a CMVpromoter and transfected into CHO cells using Effectene (Qiagen)transfection reagent according to the suppliers instructions.

[0139] Following growth of cells for 24 hours cell lysates were preparedfrom transfected and non-transfected control cells by sonication inphosphate buffered saline. Nitroreductase activity was determined byaddition of 2 μM Cy5Q and incubating at room temperature for 90 minutesfollowed by measurement of fluorescence in a CytoStar (PerSeptiveBiosystems) plate reader (FIG. 9) using 610/20 nm excitation and 670/40nm emission filters. Results showed a strong increase in fluorescence inlysate samples from nitroreductase expressing cells which wasproportional to the amount of cell lysates assayed.

EXAMPLE 6

[0140] Measurement of Cellular Nitroreductase Activity Using CellPermeable Fluorescence Substrates.

[0141] Two ethyl-ester derivatives of nitro-quenched cyanine dyes,Cy3Qee (Formula IIIa) and Cy5Qee (Formula IIIb) were synthesised.

[0142] Preparation of 5-(Carboxvmethyl)-1,2,3,3-tetramethyl-3H-indoliumIodide (1)

[0143] Methyl iodide (3 ml, 48.19 mmol) was added to a solution of2,3,3-trimethyl-3H-indol-5-yl-acetic acid (2.5 g, 11.52 mmol) insulfolan (15 ml). The reaction was heated at 48° C. for 18 hours, thencooled to room temperature. The crude reaction mixture was addeddropwise to an excess of diethyl ether and the precipitate was collectedby filtration and dried in vacuo to obtain the product as a beige solid(3.27 g, 79% yield). ¹H NMR (d₆-DMSO): δH 7.85 (d, 1H), 7.70 (s, 1H),7.50 (d, 1H), 3.95 (s, 3H), 3.75 (s, 2H), 2.75 (s, 3H), 1.50 (s, 6H).MALDI-TOF, m/z 232 (M⁺=232 for C₁₄H₁₈NO₂).

[0144] Preparation of1-(3,5-dinitrobenzyl)-5-(carboxymethyl)-3H-indolium Bromide (2)

[0145] 3,5-Dinitrobenzyl chloride (12.46 g, 57.5 mmol) was added to asolution of 2,3,3-trimethyl-3H-indol-5-yl-acetic acid (2.5 g, 11.5 mmol)with sodium bromide (5.92 g, 57.5 mmol) in sulfolan (10 ml). Thereaction was heated at 100° C. for 20 hrs, then cooled to roomtemperature. The crude reaction mixture was added dropwise to an excessof ethyl acetate and the dark brown solid was filtered OFF and dissolvedin dimethylsulfoxide before purification by reverse phasechromatography. The product was isolated as beige solid (54% yield). ¹HNMR (d₆-DMSO): δH: 8.75 (s, 1H), 8.45 (s, 2H), 7.15 (s, 1H), 6.95 (d,1H), 6.70 (d, 2H), 5.10 (s, 2H), 3.95 (s, 2H), 3.45 (s, 3H), 1.40 (s,6H). MALDI-TOF, m/z=398 (M⁺=398 for C₂₀H₂₀N₃O₆).

[0146] Preparation of5-(Carboxymethyl)-2-{(1E,3E)-5-[6-(carboxvmethyl)-1,1,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-(3.5-dinitrobenzyl)-3,3-dimethyl-3H-indoliumSalt (3).

[0147] 1,2,3,3-tetramethyl-3H-indolium iodide (1) (50 mg, 0.139 mmol)and 1-(3,5-dinitrobenzyl)-5-(carboxymethyl)-3H-indolium bromide (2) (66mg, 0.139 mmol) were dissolved in acetic acid (3.15 ml), pyridine (3.15ml) and acetic anhydride (0.7 ml) with malonaldehydebis(phenylimine)monohydrochloride (358 mg, 1.39 mmol). The reaction was heated at 70° C.for 2 hrs. The solution was then added dropwise to an excess of diethylether and the blue solid was filtered off and purified by reverse phasechromatography (HPLC with water/0.1% trifluoroacetic acid andacetonitrile/0.1% trifluoroacetic acid as eluent). The product wasisolated as a dark blue powder (33.3 mg, 31% yield). UV_(max) (H₂O)=646nm. ¹H NMR (d₆-DMSO): δH 8.75 (s, 1H), 8.45 (s, 2H), 8.35 (m, 2H), 7.5(m, 6H), 6.55 (m, 2H), 6.25 (d, 1H), 5.6 (s, 2H), 3.7 (m, 7H), 1.8 (s,3H), 1.7 (s, 2H). FAB⁺: m/z=665 (M⁺=665 for C₃₇H₃₇N₄O₈).

[0148] Preparation of1-(3,5-dinitrobenzyl)-5-(2-ethoxy-2-oxoethyl)-2-{(1E,3E)-5-[6-(2-ethoxy-2-oxoethyl)-1,1,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-3,3-dimethyl-3H-indoluimSalt (4) (Formula IIIa).

[0149]5-(Carboxymethyl)-2-{(1E,3E)-5-[6-(carboxymethyl)-1,1,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-3H-indolium(3) (4.5 mg, 0.0058 mmol) was dissolved in ethanol (2 ml) withconcentrated hydrochloric acid (2011) and stirred at room temperatureunder nitrogen for 16 hrs. The solvent was removed under reducedpressure and the residue purified by reverse phase chromatography (HPLCwith water/0.1% trifluoroacetic acid and acetonitrile/0.1%trifluoroacetic acid as eluent) to isolate the product as a blue solid(4.1 mg, 85% yield). UV_(max) (H₂O)=647 nm. ¹H NMR (CDCl₃): δH 8.95 (s,1H), 8.4 (s, 2H), 7.85 (m, 2H), 7.3 (m, 6H), 6.85 (d, 1H), 6.5 (m, 2H),5.5 (s, 2H), 4.2 (q, 4H), 3.7 (s, 4H), 3.65 (s, 3H), 1.8 (s, 3H), 1.7(s, 3H), 1.25 (t, 6H). FAB⁺: m/z=721 (M⁺=721 for C₄₁H₄₆N₄O₈).

[0150] Preparation of5-{2-[(acetyloxy)methoxy]-2-oxoethyl}-2-[(1E,3E)-5-(6-{2-[(acetyloxy)methoxy]-2-oxoethyl}-1,1,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)-1,3-pentadienyl]-1-(3,5-dinitrobenzyl)-3.3-dimethyl-3H-indoliumSalt (5).

[0151] To a solution of5-(Carboxymethyl)-2-{(1E,3E)-5-[6-(carboxymethyl)-1,1,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene]-1,3-pentadienyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-3H-indolium(3) (10 mg, 0.015 mmol) in anhydrous acetonitrile (2 ml) withN,N-diisopropylethylamine (4.7 mg, 0.0375 mmol) was addedbromomethylacetate (23 mg, 0.150 mmol). The reaction was stirred at roomtemperature under a nitrogen atmosphere for 24 hours. The solvent wasthen evaporated under reduced pressure and the blue residue was purifiedby reverse phase chromatography (HPLC). The product was isolated as ablue powder (10.6 mg, 83% yield). UV_(max) (H₂O)=643 mm. ¹H NMR (CDCl₃):δH 9.05 (s, 1H), 8.5 (s, 2H), 8.35 (m, 1H), 7.35 (m, 6H), 7.2 (m, 1H),7.0 (m, 1H), 6.45 (m, 1H), 6.15 (m, 1H), 5.75 (s, 2H), 3.75 (s, 3H), 3.7(s, 4H), 2.1 (s, 3H), 1.05 (s, 3H), 1.75 (s, 6H), 1.70 (s, 6H). FAB⁺:m/z=809 (M⁺=809 for C₄₃H₄₅N₄O₁₂).

[0152] Preparation of5-(Carboxymethyl)-2-{(1E)-3-[6-(carboxymethyl)-1,1,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene]-1-propenyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-3H-indoliumSalt (6).

[0153] 1,2,3,3-tetramethyl-3H-indolium iodide (1) (100 mg, 0.28 mmol)and 1-(3,5-dinitrobenzyl)-5-(carboxymethyl)-3H-indolium bromide (2) (133mg, 0.28 mmol) were dissolved in acetic acid (2.25 ml), pyridine (2.25ml) and acetic anhydride (0.25 ml) with N,N′-diphenylformamidine (55 mg,0.28 mmol). The reaction was heated at 70° C. for 2 hrs. The solutionwas then added dropwise to an excess of diethyl ether and the red solidwas filtered then purified by reverse phase chromatography (HPLC withwater/0.1% trifluoroacetic acid and acetonitrile/0.1% trifluoroaceticacid as eluent). The product was isolated as a dark pink powder (16.5mg, 8%yield). UV_(max) (H₂O)=553 nm. MALDI-TOF: m/z=640 (M⁺=639 forC₃₅H₃₅N₄O₈).

[0154] Preparation of1-(3,5-dinitrobenzyl)-5-(2-ethoxy-2-oxoethyl)-2-{(1E)-3-[6-(2-ethoxy-2-oxoethyl)-1,1,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene]-1-propenyl}-3,3-dimethyl-3H-indoluimSalt (7) (Formula IIIb).

[0155]5-(Carboxymethyl)-2-{(1E)-3-[6-(carboxymethyl)-1,1,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene}-1-propenyl}-1-(3,5-dinitrobenzyl)-3,3-dimethyl-3H-indolium(6) (4 mg, 0.00531 mmol) was dissolved in ethanol (2 ml) withconcentrated hydrochloric acid (50 μl) and stirred at room temperatureunder nitrogen for 20 hrs. The solvent was removed under reducedpressure and the residue was purified by reverse phase chromatography(HPLC with water/0.1% trifluoroacetic acid and acetonitrile/0.1%trifluoroacetic acid as eluent) to isolate the product as a pink solid(3.2 mg, 74% yield). UV_(max) (H₂O)=549 nm. ¹H NMR (CDCl₃): δH 9.0 (s,1H), 8.4 (s, 2H), 8.45 (m, 1H), 7.3 (m, 6H), 6.9 (d, 1H), 6.45 (m, 1H),5.6 (s, 2H), 4.2 (q, 4H), 3.75 (s, 4H), 3.7 (s, 3H), 1.85 (s, 3H), 1.7(s, 3H), 1.3 (t, 6H). MALDI TOF: m/z=696 (M⁺=695 for C₃₉H₄₃N₄O₈).

[0156] Cy3Qee (Formula IIIa) and Cy5Qee (Formula IIIb) were evaluated ascell permeable fluorescence substrates for nitroreductase measurement inliving cells. Nitroreductase expressing cells and control cells werecultured at 10,000 cells/well in 96 well plates in tissue culture mediumcontaining 10% foetal calf serum and incubated at 37° C. in the presenceof 101M Cy3Qee or 30 μM Cy5Qee. Fluorescence measurements were madeusing a CytoStar (PerSeptive Biosystems) plate reader using 530/25 nmexcitation and 580/50 nm emission filters for Cy3Qee and 610/20 nmexcitation and 670/40 nm emission filters for Cy5Qee.

[0157] Fluorescence measurements showed a significant time dependentincrease in fluorescence in nitroreductase expressing cells with minimalchanges in fluorescence in control cells indicating that both Cy3Qee(FIG. 10) and Cy5Qee (FIG. 11) are effective cell permeable substratesfor measurement of nitroreductase activity in living cells.

EXAMPLE 7

[0158] Measurement of Cellular Nitroreductase Activity in Living Cellsby Flow Cytometry.

[0159] Nitroreductase expressing cells and control cells were incubatedfor 2 hours at 37° C. in tissue culture media containing 30 μM Cy3Qee.Following incubation cells were washed with phosphate buffered salineand trypsinised to produce cell suspensions for flow cytometry.

[0160] Cells were analysed using a FACScalibur flow cytometer (BectonDickinson) using 488 nm laser excitation and a 585/42 nm emissionfilter. Results (FIG. 12) show a significant increase in fluorescence innitroreductase expressing cells (mean fluorescence 169.9) compared withcontrol cells (mean fluorescence 18.2).

EXAMPLE 8

[0161] Preparation of Cy5™-Cascade Blues Ester Linked Cassette

[0162] Cy5Q mono free acid potassium salt (obtained from AmershamPharmacia Biotech Ltd) (5 mg, 0.006 mmol), Cascade blue 0 (MolecularProbes) (8-hydroxypyrene-1,3,6-trisulfonic acid sodium salt (8.8 mg,0.018 mmol) (Fluka)), N, N diisopropylcarbodiimide (10 μl, 0.065 mmol),1-hydroxybenzotriazole (1 mg, 0.007 mmol), 4-(dimethylamino)pyridine(0.9 mg, 0.007 mmol) and activated molecular sieves powder (250 mg) werestirred together in anhydrous N,N-dimethylformamide (2 ml) at roomtemperature for 12 hrs. A new product spot was observed by TLC (RP C₁₈1:1 MeOH:Water), rf=0.8 (compared to free Cy5Q; rf=0.72). The molecularsieves were filtered off and the product precipitated into diethylether. The precipitate was filtered off, washed with ethyl acetate anddried. The product was purified by RP C₁₈ flash column chromatography.Unreacted 8-hydroxypyrene-1,3,6-trisulfonic acid sodium salt was elutedfrom the column with water and the product then eluted with 5%acetonitrile/water. Fractions containing pure product were collected andthe majority of the solvent removed under reduced pressure. The residuewas freeze dried to give the product as a cyan powder (1 mg, 11%).

[0163] λmax (Water) 648 nm (Cy5Q) 354, 372 nm (pyrene).

[0164] MALDI-TOF MS; found 1246 (MH⁺); [theoretical (C₅₄H₄₄N₄O₂₁S₅)1245].

[0165] Fluorescence; Cy5Q-Cascade blue ester linked cassette hasnegligible emission at 510 nm (FIG. 13); 510 nm is the emission maximafor free 8-hydroxypyrene-1,3,6-trisulfonic acid sodium salt in water,(FIG. 14). This indicates efficient energy transfer from Cascade blue toCy5Q and quenching of the fluorescence of the Cascade blue moiety whenbound to Cy5Q within the cassette. When treated with sodium hydroxidesolution, fluorescence emission at 510 nm is observed (FIG. 15),indicating the chemical hydrolysis of the ester linkage and theliberation of the fluorescent 8-hydroxypyrene-1,3,6-trisulfonic moiety.

[0166] In vitro Evaluation

[0167] Cy5Q-Cascade blue cassette (0.8 mM in acetate buffer pH 5.0) wasdiluted to 811M in PBS pH 7.4 containing 1 mM NADH. A cell lysate wasprepared from 2×10⁷ SKOV cells by re-suspension of scraped cells in 5 mlPBS at 4° C. and repeated passage through a 25 gauge syringe needle.Aliquots (1 ml) of Cy5Q-Cascade blue were incubated with 500111 of celllysate or PBS for 20 minutes at 37° C. Following incubation 100 μl of a1 ng/μl solution of E. coli B nitroreductase was added to one sample andincubation continued for 5 minutes. Aliquots (100 ml) were thendispensed in quadruplicate from each sample to a 96 well plate formeasurement of fluorescence on a Cytofluor plate reader using 450 nmexcitation/530 nm emission filters for cascade blue and 610 nmexcitation/670 nm emission for Cy5.

[0168]FIG. 16 shows an increase of fluorescence emission at 530 nm (i.e.of Cascade Blue) when the cassette is incubated in the presence of celllysate and an increase in fluorescence emission at 670 nm (i.e. Cy5)when nitroreductase enzyme is present.

1 A method for increasing the fluorescence of a dye molecule comprisingat least one NO₂ group characterised by the reduction of said at leastone NO₂ group to NHOH or NH₂. 2 A method as claimed in claim 1 whereinthe a dye molecule comprising at least one NO₂ group is a cyanine dye. 3A method according to claim 2 wherein the cyanine dye moleculecomprising at least one NO₂ group has Formula I:

wherein groups R³, R⁴, R⁵ and R⁶ are attached to the rings containing Xand Y or, optionally, are attached to atoms of the Z¹ and Z² ringstructures and n is an integer from 1-3; Z¹ and Z² each represent a bondor the atoms necessary to complete one or two fused aromatic rings eachring having five or six atoms, selected from carbon atoms and,optionally, no more than two oxygen, nitrogen and sulphur atoms; X and Yare the same or different and are selected from bis-C₁-C₄ alkyl- andC₄-C₅ spiro alkyl-substituted carbon, oxygen, sulphur, selenium, —CH═CH—and N—W wherein N is nitrogen and W is selected from hydrogen, a group—(CH₂)_(m)R⁸ where m is an integer from 1 to 26 and R⁸ is selected fromhydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl,hydroxyl, sulphonate, sulphate, carboxylate, phosphonate, polyethyleneglycol, substituted amino, quaternary ammonium, nitro, primary amide,substituted amide, and groups reactive with amino, hydroxyl, carbonyl,carboxyl, phosphoryl, and sulphydryl groups; groups R³, R⁴, R⁵, R⁶ andR⁷ are independently selected from the group consisting of hydrogen,substituted or unsubstituted C₁-C₄ alkyl, OR⁹, COOR⁹, nitro, amino,acylamino, quaternary ammonium, phosphate, sulphonate and sulphate,where R⁹ is substituted or unsubstituted and selected from H, C₁-C₄alkyl, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl,hydroxyl, sulphonate, sulphate, carboxylate, substituted amino,quaternary ammonium, nitro, primary amide, substituted amide, and groupsreactive with amino, hydroxyl, carbonyl, carboxyl, phosphoryl, andsulphydryl groups; groups R¹ and R² are selected from C₁-C₁₀ alkyl whichmay be unsubstituted or substituted; characterised in that at least oneof the groups R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ comprises at least one nitrogroup which reduces the fluorescence emission of said dye such that itis essentially non-fluorescent. 4 A method according to claim 2 or 3wherein the cyanine dye molecule comprising at least one NO₂ group is acompound of Formula II or Formula III, or salts thereof.

5 A method according to any of claims 1 to 4 wherein reduction of saidat least one NO₂ group is catalysed by an enzyme. 6 A method accordingto claim 5 wherein reduction of said at least one NO₂ group is catalysedby nitroreductase and, preferably, bacterial nitroreductase. 7 A methodfor detecting nitroreductase enzyme activity in a compositioncomprising: a) mixing said composition with a cyanine dye moleculecomprising at least one NO₂ group under conditions to promotenitroreductase activity; and b) measuring an increase in fluorescencewherein the increase is a measure of the amount of nitroreductaseactivity. 8 A method according to claim 7 wherein the compositioncomprises a cell or cell extract. 9 A method of detecting analytescomprising: a) providing a nitroreductase enzyme coupled to an assayreagent under conditions where the amount of activity of the enzyme isproportional to the amount of analyte in the assay; b) providing acyanine dye molecule comprising at least one NO₂ group; and c) measuringan increase in fluorescence as a measure of the amount of nitroreductaseactivity. 10 An assay method which comprises: a) binding a firstcomponent of a specific binding pair to a surface; b) adding a secondcomponent of the binding pair under conditions to promote bindingbetween the components, said second component being labelled with anitroreductase enzyme; c) adding a cyanine dye molecule comprising atleast one NO₂ group under conditions suitable for nitroreductaseactivity; and d) detecting binding of the second component to the firstcomponent by measuring an increase in fluorescence as a measure of boundnitroreductase activity. 11 A method according to claim 10 wherein saidspecific binding pair is selected from the group consisting ofantibodies/antigens, lectins/glycoproteins, biotin/streptavidin,hormone/receptor, enzyme/substrate, DNA/DNA, DNA/RNA and DNA/bindingprotein. 12 An assay method which comprises: a) contacting a host cellwith a cyanine dye molecule comprising at least one NO₂ group, whereinsaid host cell has been transfected with a nucleic acid moleculecomprising expression control sequences operably linked to a sequenceencoding a nitroreductase; and b) measuring an increase in fluorescenceas a measure of nitroreductase gene expression. 13 An assay methodaccording to claim 12 wherein the cyanine dye molecule is permeable tocells. 14 An assay method as claimed in claim 12 or 13 wherein thecyanine dye molecule is a compound of Formula I. 15 An assay methodaccording to any of claim 14 wherein at least one of R¹ to R⁷ of thecyanine dye molecule of Formula I comprises a cell membranepermeabilising group. 16 A cyanine dye molecule of Formula I comprisingat least one NO₂ group characterised in that said cyanine dye moleculeis cell permeable. 17 A cyanine dye molecule as claimed in claim 16wherein said cyanine dye molecule is selected from compounds of FormulaIIIa or Formula IIIb, or salts thereof.

18 An assay method which comprises: a) contacting a host cell extractwith a cyanine dye molecule comprising at least one NO₂ group whereinsaid host cell has been transfected with a nucleic acid moleculecomprising expression control sequences operably linked to a sequenceencoding a nitroreductase; and b) measuring an increase in fluorescenceas a measure of nitroreductase gene expression. 19 A method according toany of claims 1 to 15 or 18 wherein the increased fluorescence of thecyanine dye molecule is measured by analysis of fluorescence emission inthe range 500-900 nm. 20 A method according to claim 19 wherein theincreased fluorescence of the cyanine dye molecule is measured byanalysis of fluorescence emission in the range 665 nm to 725 nm. 21 Akit for a reporter system comprising a means for expressing anitroreductase enzyme and a cyanine dye molecule comprising at least oneNO₂ group. 22 A kit for detecting the presence of one component of aspecific binding pair comprising a nitroreductase enzyme coupled to theother component of said specific binding pair and a cyanine dye moleculecomprising at least one NO₂ group. 23 A paired fluorophore ratiometricreporter of Formula IV:

wherein: D₁ is a detectable fluorophore; D₂ is a cyanine dye moleculehaving Formula (1); and L is a linker group. 24 A paired fluorophoreratiometric reporter of claim 23 wherein said reporter is membranepermeable. 25 A paired fluorophore ratiometric reporter of claim 23 orclaim 24 wherein L is a cleavable linker group.
 26. An assay methodaccording to any of claims 9 to 15 comprising the paired fluorophoreratiometric reporter of any of claims 23 to 25.